The potential uses of κ-carrageenase is to obtain low molecular weight carbohydrates, for kelp digestion, for prevention of biofouling by controlling red algal bloom formation, to obtain fine chemicals and for algal biotechnology.
Carrageenan is a natural ingredient obtained from certain species of the red seaweed; class Rhodophyceae (Greer, C. W., and W. Yaphe. 1984. Enzymatic analysis of carrageenans: Structure of carrageenan from Eucheuma nudum. Hydrobiologia. 116/117: 563-567). Carrageenan is a linear polysaccharide made up of repeating dissacharide sequence of 1,3 linked β-D-galactopyranose called the A residue and α-D-galactopyranose residues linked through positions 1,4 called the B residue. Carrageenans are distinguished from agars in that the B units in carrageenan are in the D form whereas they are in the L form in agar. The regular backbone of the basic structure of carrageenan is disrupted by a more or less ordered distribution of sulfate hemi ester groups. Carrageenan can also contain some methoxy and pyruvate groups. Popular sources for carrageenan are Chondrus, Eucheuma (Kappaphycus), Gigartina and Iridaea species. Three basic types of carrageenan are available. Kappa (κ), iota (ι) and lambda (λ) carrageenan which are obtained from Chondrus, Eucheuma and Gigartina species, which differ in the number and location of sulfate ester substitution. κ and ι-carrageenan form thermally reversible gels in the presence of K+, Ca+2 or NH4+ but do not gel in the Na+ form.
Hydrolases, which degrade carrageenans at β-1,4 linkages, are known as carrageenases. Three types of enzymes, viz. κ, ι and λ-carrageenases, have been isolated from various marine bacteria. They all are endohydrolases that cleave the β-1,4 linkages of carrageenans yielding products of the neocarrabiose series (Michel, G., L. Chantlat, E. Fanchon, B. Henrissat, B. Kloareg, and O. Didweberg. 2001. The ι-carrageenase of Alteromonas fortis. Journal of Biological Chemistry. 276(43): 40202-40209). κ-carrageenase has large-scale application and industrial demand in the forth-coming years (Ostgaard, K., B. F. Wangen, S. H. Knutsen, and I. M. Aasen. 1993. Large scale production and purification of κ-carrageenase from Pseudomonas carrageenovora for applications in seaweed biotechnology. Enzyme and Microbial Technology. 15(4): 326-333).
Jhonston and McCandless in a paper entitled “Enzymic hydrolysis of the potassium chloride soluble fraction of carrageenan: properties of λ-carrageenases from—Pseudomonas carrageenovora” in Canadian Journal of Microbiology 19: (1973) p.p. 779-788, has reported that carrageenase from Pseudomonas carrageenovora exhibited activity only against KCl soluble fraction of carrageenan i.e. λ-carrageenan. They could improve the yield of λ-carrageenase but could not totally eliminate simultaneous production of κ-carrageenase. Also, after using multiple and complicated steps of purification, specific activity achieved was only 7 galactose units/mg protein which had temperature and pH optima 28° C. and 6.2 respectively. The drawback of this work is that carrageenase is active only in acidic condition and at 28° C. temperature. Hence it can not be used in alkaline condition as well as at elevated temperature. Moreover, the process did not yield noteworthy purification of enzyme, in spite of using complicated enzyme purification steps.
Bellion et al. in a paper entitled “The degradation of Eucheuma spinosum and Eucheuma cottoni carrageenans by ι and κ-carrageenases from marine bacteria” in Canadian Journal of Microbiology 28(7): (1982) p.p. 874-880 has reported the degradation of Eucheuma spinosum and Eucheuma cottonii carrageenans by ι-carrageenase and κ-carrageenase from marine Pseudomonas carrageenovora and identified the hydrolyzed products. The bacterium was cultivated in a medium consisting of g. I−1 NaCl 25; K2HPO40.1; MgSO47H2O 5.0; CaCl22H2O 0.2; casamino acids 2.5; carrageenan 2.5; 0.3% FeSO4 (10 ml/lit) and optimum incubation temperature of 22° C. The drawback is that the medium used for the production of carrageenase by this bacterium consisted of 7 components. Moreover, optimum incubation temperature used was 22° C., which is not feasible for the cultivation of this type of bacteria in temperate countries without cooling device especially in summer.
Sarwar et al. in a paper entitled “Potentiality of artificial sea water salts for the production of carrageenase by a marine Cytophaga species” in Microbiology and Immunology. 29(5): (1985) p.p. 405-411 and “Purification of a κ-carrageenase from marine Cytophaga species” in Microbiology and Immunology. 31(9): (1987) p.p. 869-877 wherein, 8 components were used in the medium for the production of carrageenase by a marine Cytophaga sp. Moreover, this culture required a suitable combination of NaCl and MgCl2 for carrageenase production. The pH and temperature optima were 7.6 and 25° C. respectively. The activity achieved after following complicated steps of purification was only 5.0 galactose units/mg protein. The drawback of this work is that the presence of NaCl and MgCl2 in the medium is a must for carrageenase production and inspite of using eight medium components and complicated steps of purification; only 5.0 galactose units/mg protein was obtained as enzyme activity. This low yield makes the enzyme production commercially uneconomical.
Fleurence et al. in a paper entitled “Use of enzymatic cell wall degradation for improvement of protein extraction from Chondrus crispus, Gracilaria verrucosa and Palmaria palmata” in Journal of Applied Phycology 7: (1995) 393-397) wherein, κ-carrageenase was reported to have maximum activity between pH 6.5-6.8 and at 45° C. and lower activity at acidic and alkaline conditions. The drawback of this work is that the enzyme is active only at neutral pH hence can not be used either in acidic and alkaline conditions. Moreover, poor activity at lower temperature makes its applicability limited to higher temperature.
Dyrset et al. in a paper entitled “Development of a fermentation process for production of a κ-carrageenase from Pseudomonas carrageenovora” in Enzyme and Microbial Technology 20(6): (1997) p.p. 418-423 has reported a fermentation process for the production of κ-carrageenase using two strains of Pseudomonas carrageenovora. The medium used for this study contained gl−1 of CaCl2, 2H2O 0.2; Casamino acid 6.8; KCl 0.3; K2HPO43.0; MgSO4, 7H2O 0.5; NaCl 20.0; NH4Cl 0.7; (NH4)2 SO4 5.0; Carrageenan 2.5. The pH of the medium was 7.0 and temperature was 20° C. The drawback of this work is that large number of components as well as high substrate concentration is required in the medium and the maintenance of pH 7.0 with all these components is difficult. Besides, the fermentation process is carried out at 20° C., which requires special device for maintaining such low temperature
Araki et al. in a paper entitled “Purification and characterization of κ-carrageenase form a marine bacterium Vibrio sp. CA-, 1004” in Fisheries Science 65 (6): (1999) p.p. 937-942 wherein, they purified and characterized κ-carrageenase, an inducible enzyme, from a marine bacterium Vibrio species, the molecular weight of which was 35 KDa and maximum enzyme activity at pH 8.0 and temperature 40° C. The medium used for this study contained g 1−1 of peptone 5.0; yeast extract 1.0; NaCl 30; MgSO4 0.5; K2HPO4 2.0; KH2PO4 0.4; carrageenan 15. The activity of crude carrageenase obtained was 0.949 galactose units/mg protein. The drawback of this work is that production medium containing seven components yielded carrageenase with activity of only 0.949 galactose units/mg protein. Such low activity would invariably make the process uneconomical and unfeasible.
Japanese patent No. JP2001136961 (2001) assigned to Okita Yuji et al. has disclosed a “Method for controlling carrageenase producing ability of bacterium” is described. Here, a method has been described to control the carrageenase producing ability of a carrageenase producing bacterium, by culturing it in the presence of a bacterium free from the carrageenase producing ability or its culture product. The drawback of this invention is that production of carrageenase was controlled only by co-cultivating carrageenase producer with that of carrageenase non producer.
Japanese patent No. JP 2000116376 (2000) assigned to Araki Toshiyoshi entitled “New κ-carrageenase, microorganism for producing the same, production of carrageenase and its use” has disclosed that κ-carrageenase has decomposition activity against κ-carrageenan. This enzyme decomposed κ-carrageenan into neocarrabiose and neocarratetraose and had pH optima of 8.0. The drawback of this invention is that the enzyme had only alkaline pH optima hence can not be used in acidic condition.
Japanese patent No. JP1006656 (1998) assigned to Christian G et al. entitled “Production of κ-carrageenase” has disclosed the productivity of κ-carrageenase by Pseudomonas carrageenovora, Alteromonas or Cytophoga which was substantially improved by controlling the pH of a culture medium by nitrogen containing base (ammonium water) to be assimilated by these bacteria. By this, κ-carrageenase production could be improved from >=20 galactose Units/ml to 40 to 60 galactose Units/ml. The drawback of this invention is that they could achieve only 3fold purification after controlling pH which is not viable commercially.
Canadian Patent No. CN1544623 (2004) assigned to Haigin M et al. entitled “Carrageenin catabolic enzymes it's preparing process and application”, wherein, an enzyme that can degrade κ-carrageenan to prepare oligocarrageenan and degrade beta-1,4 indican bond of κ-carrageenan having a molecular weight of 30 KDa was obtained. Cytophaga sp. was cultured at 28-35 deg. C., centrifuged to obtain crude enzyme and concentrated it by using 40-80% ammonium sulphate. This method was compared with chemical method; it had simple preparing course, high product yield, and stable quality and ensure the activity research and development of oligosaccharide etc. The drawback of the present invention is that only salting out method was used for concentrating enzyme which does not give substantially concentrated/purified enzyme preparation.